It has been desirable to develop materials suitable for forming water-insoluble organic thin films which can fix biologically active proteins inside the materials thereof for use as biosensors, such as enzyme electrodes. There are only a few types of organic thin films utilized to date. For example, those prepared by dispersing a biologically active protein into resin materials and hardening the resin materials through optical exposure in the presence of a light-sensitive cross-linking agent, e.g., as disclosed in JP-A-62-237348, JP-A-62-238453, JP-A-62-50656, JP-A-6l-l53559 and EP 0214805 (The term "JP-A" as used herein means an "unexamined published Japanese patent application"). In these films, water-soluble polymers, such as polyvinyl alcohol, polyvinyl pyrrolidone and the like, are used as the resin materials. The cross-linking agents used are dichromic acid, diazo compounds, diazide compounds and so on. The hardening is effected by cross-linking under irradiation with ultraviolet rays.
Additionally, there are films prepared by mixing bioprotein, such as albumin, or a resin material with a biologically active protein, and making the mixture into a film through cross-linking in the presence of a dialdehyde such as gluteraldehyde. Such a film forming method has been widely used because of its simplicity, and instances of enzyme sensors prepared utilizing this method are cited in JP-A-62-23554, JP-A-56-88794,JP-A-62-240849, WO 8607632, EP 0230472 and so on.
Organic thin films formed by the above-described methods are moderately sturdy. In the former case, however, it is feared that the irradiation with light, such as ultraviolet rays, tends to inactivate the biologically active proteins, particularly antibodies. In the latter case, glutaraldehyde is a strong reacting agent and reacts directly with the biologically active protein molecules themselves so as to cross-link them. Thus, the biologically active protein itself is deactivated in part by the film formation and this tends to cause a drop in operability of the sensor or the like.
Also, films disclosed, e.g., in JP-A-6l-23l454 and JP-A-62-l83882 have similar problems because of the analogous hardening means taken therein.
In addition, a method of forming films by covalently bonding succimidyl acrylate to enzyme molecules, in advance, and copolymerizing the reactive monomer obtained and other monomers in the presence of an initiator is disclosed in Analytical Chemistry, vol. 57, p. 1920 (1985). However, in analogy with the above-cited instances, there is a fear that the polymerization reaction tends to inactivate the active protein because the polymerization is carried out in the presence of the biologically active protein.
As a result, it has been desired to improve the sensitivity of a biosensor by developing organic thin films, other than the above-described ones, and preparation methods thereof, which are not only excellent in film formability and strength, but also almost free from inactivation of biologically active protein.